Normal gastrointestinal motility requires maintenance of enteric nerves and the networks of interstitial cells of Cajal (ICC). The overall objective of this proposal is to identify the mechanisms that underlie the pathobiology of diabetic gastroenteropathy (DGE). Our preliminary data suggest a central role for the neuronal nitric oxide synthase/nitric oxide (nNOS/NO) and the heme oxygenase/carbon monoxide (HO/CO) pathways in the maintenance of ICC networks in diabetes. We have made the following key preliminary observations: HO1 is up-regulated in diabetes and serves as a protective mechanism against diabetes-induced oxidative stress. NO up-regulates HO1. Loss of nNOS is an early step in the development of DGE resulting in decrease in NO, loss of up-regulation of HO1 and subsequent decrease in ICC numbers. Supporting the hypothesis that NO is cytoprotective to ICC are preliminary data that show that ICC numbers are decreased in nNOS knockout mice, NOS inhibitors decrease ICC numbers, and NO donors increase ICC numbers in culture. Also, nNOS expression is decreased, HO1 up-regulation is lost and ICC numbers are decreased in patients with DGE. We have also developed animal and human tissue models for DGE and developed a non-invasive test to determine gastric emptying in mice. Based on this work we have generated the novel hypothesis that up-regulation of the HO1/CO pathway by reactive oxygen species serves as an initial cytoprotective mechanism against damage to ICC by AGE and reactive oxygen species. Loss of nNOS expression from enteric nerves leads to loss of continued up-regulation of HO1 resulting in oxidative injury to ICC. Loss of ICC results in loss of the pacemaker signal, failure to amplify neuronal signals and dysmotility. The proposal has two specific aims. In the first aim we will test the hypothesis that DGE results from initial loss of nNOS expression and subsequent loss of ICC. In the second aim we will test the hypothesis that the HO/CO pathway is cytoprotective to ICC and that aberrant regulation of the HO/CO pathway results in decreased ICC and subsequent DGE. The studies will employ newly developed gastric emptying and ICC culture techniques, electrophysiology, immunohistochemistry, and molecular biology to answer the questions raised. We are now uniquely poised to provide new information on the mechanisms that underlie the development of DGE. As this work will identify basic mechanisms that underlie maintenance of ICC it also has implications for further understanding the pathophysioIogy of many of the motility disorders linked to disorders in ICC number and function.